Licheng Wu

It is generally reasoned that lethal infections caused by opportunistic pathogens develop permissively by invading a host that is both physiologically stressed and immunologically compromised. However, an alternative hypothesis might be that opportunistic pathogens actively sense alterations in host immune function and respond by enhancing their virulence phenotype. We demonstrate that interferon-gamma binds to an outer membrane protein in Pseudomonas aeruginosa, OprF, resulting in the expression of a quorum-sensing dependent virulence determinant, the PA-I lectin. These observations provide details of the mechanisms by which prokaryotic organisms are directly signaled by immune activation in their eukaryotic host.

Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13-induced, claudin-2-dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.

Severe and prolonged states of catabolic stress have been shown to have profound effects on the intestinal tract microflora and intestinal function. Gut-derived sepsis is a term used to describe a state of systemic inflammation with organ dysfunction after severe catabolic stress hypothesized to be initiated and perpetuated by the intestinal tract microflora. Popular notions of the mechanism of this process have suggested that stress promotes the translocation of intestinal bacteria or their toxins into the systemic compartment resulting in the release of proinflammatory cytokines which participate in the systemic inflammatory response syndrome. This review is an attempt to redefine the mechanism of gut-derived sepsis by focusing on molecular events that result from host-pathogen interactions within the intestinal tract itself. This evidence-based review posits that gut-derived bacteremia, even with potent nosocomial pathogens, is an event of low proinflammatory potential and, itself, is an insufficient stimulus for the systemic inflammatory response and organ failure state typically seen after severe and prolonged catabolic stress. Mechanisms of this apparent paradox are discussed.

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.